Understanding the mechanisms of an effective neutralizing antibody response to HIV is one of the highest priorities in the field of HIV-specific immunity. In this regard, the inability of the humoral response of most vaccinees to cross-neutralize multiple strains of HIV is believed to be a major obstacle to the design of effective vaccines. In 2006 we observed that sera from subpopulation of our chronically infected cohorts had considerable neutralization breadth extending across clades. Much of the work done prior to that time had not focused on patients selected for broadly cross-neutralizing antibodies to HIV-1. In addition, a relatively small number of monoclonal neutralizing antibodies existed at the time. For these reasons we recruited a cohort of individuals screened for broadly cross-neutralizing antibodies to HIV. We, in collaboration with investigators at the Vaccine Research Center (VRC), used these sera to systematically dissect the means by which these patients cross-neutralize HIV-1. We thus far have identified 30 such patients and are continuing to accrue additional subjects. A number of fundamental questions had not been addressed with regard to the HIV-specific humoral immune response of these patients. For example, it was not known if these patients had genetic or clinical characteristics, or HIV-specific cellular immune response characteristics in common. Given that our patients are infected with clade B viruses and should not have experienced infection by viruses belonging to multiple clades, we hypothesized that cross-neutralization is mediated through conserved epitopes on HIV envelope (Env). In addition, although considerable work had been done on patient sera, very little had been done on HIV-specific B cells. The phenotype and immunoglobulin class of HIV-specific B cells in comparison to responses to other viruses remained poorly defined. Further, it remained unclear whether patients with broad cross-neutralizing activity are unique with regard to these parameters. One primary objective of our work on the humoral response to HIV is to understand the basis of a broadly cross-neutralizing antibody response in our patients. It was not known whether there are common features of the humoral response of such patients with regard to specificity. It was also not known whether neutralization was mediated by a few B cell clones directed to conserved epitopes or by an extremely polyclonal response to many epitopes. To dissect the specificity and diversity of epitopes targeted by the B-cell response in patients with broad sera, we have initiated a collaborative effort to isolate monoclonal antibodies. To further understand the specificities and binding characteristics that underlie a broadly neutralizing antibody response we developed techniques that permitted isolation of human monoclonal antibodies without previous knowledge of specificity. In this technique peripheral blood memory B cells are sorted and expanded for 13 days with interleukin (IL)-2, IL-21 and CD40-ligand expressing cells. The supernatants of large numbers of micro-cultures of these cells can then be screened for neutralizing activity in a high-throughput manner. From the cultures that exhibit anti-HIV neutralizing activity the immunoglobulin genes can then be isolated, re-expressed, and characterized. In the recent past we have used these techniques to isolate antibodies with novel specificities on HIV Env that represent conserved sites of vulnerability that can be targeted in prophylaxis and immunotherapies. We previously isolated an antibody, designated 10E8, which is among the most broad thus far described. It binds the gp41 membrane-proximal external region (MPER) of Env. It neutralizes 98% of tested viruses. 10E8 also provided protection from mucosal challenge with a simian immunodeficiency virus expressing an HIV Env glycoprotein. We have also isolated an antibody termed 35O22 that neutralizes by binding a conserved face on contiguous areas of gp41 and gp120. This was one of a novel class of antibodies that bind across the gp41-120 interface. These results suggested that such broadly neutralizing antibodies can mediate potent protection in vivo and provide a rationale for use of these antibodies in prophylaxis or induction of these antibodies by vaccines to prevent HIV infection. More recently, we have isolated a CD4-binding site antibody, termed N6, that potently neutralized 98% of HIV-1 isolates, including 16 of 20 that were resistant to other members of its class. N6 evolved a mode of recognition such that its binding was not impacted by single amino acid changes across the immunoglobulin heavy chain. In addition, structural analysis revealed that N6 shifted in orientation compared to other members of its class, permitting it to avoid steric clashes with glycans, which is a common mechanism of resistance. Thus, an HIV-1-specific bNab can develop specific alterations that allow it to avoid mechanisms of HIV-1 resistance that impede other members of its class and can achieve near-pan neutralization of HIV-1. This antibody is among the lead candidates for clinical development for prophylaxis and therapy. Through collaborative work N6 and 10E8 have been further modified to either increase potency and manufacturability. Some collaborators have incorporated N6 or 10E8 sequences into bi- or tri-specific antibodies that increase the breadth or potency compared to individual antibodies. Passive administration of single, bi-, or tri-specific antibodies are in various stages of pre-clinical or clinical development for therapy or prophylaxis. Through this work we continue to provide a better understanding of the specificities and functions of the HIV-specific humoral immune response that are likely to provide protection from infection. Over the coming years we anticipate that this work, in the context of work from other groups, will greatly enhance our knowledge of what features of this response should be induced in vaccination strategies.